Polypeptide of human-origin hyaluronate synthetase and DNA encoding the same

ABSTRACT

A DNA encoding at least part of a hyaluronan synthase of human origin, particularly encoding the whole or a part of an amino acid sequence shown by SEQ ID NO: 4. A polypeptide of the hyaluronan synthase of human origin, which is encoded by the DNA, may have a substitution, deletion or insertion of one or more amino acid residues that does not substantially lower an activity of synthesizing hyaluronan. A polypeptide of the hyaluronan synthase of human origin or a part thereof encoded by the DNA is also provided.

TECHNICAL FIELD

The present invention relates to a polypeptide of a hyaluronan synthaseof human origin and a DNA encoding the same.

BACKGROUND ART

Hyaluronan is one of high molecular weight glycosaminoglycans and isconstituted by repeated β-1,4 linked disaccharide units, each of theunit being composed of glucuronic acid linked to N-acetylglucosamine bya β-1,3 bond (GlcUAβ1-3GlcNAc; GlcUA and GlcNAc represent glucuronicacid and N-acetylglucosamine, respectively). Hyaluronan is acharacteristic constituent of the extracellular matrix at the earlystage of morphogenesis of animals. Its synthesis is regulated spatiallyand temporally (Toole, B. P. (1981) Cell Biology of the ExtracellularMatrix (Hey, E. D., ed.) pp. 259-294, Plenum, New York). Accumulation ofhyaluronan on the cell surface is correlated with regulation of behaviorof cells, particularly migration, adhesion, cure of wounds, infiltrationof tumors, and the like (Turley, E. A. (1989) The Biology of Hyaluronan,Ciba Foundation Symposium 143, pp. 121-137; Wiley, Chichester, England;Knudson, W., Biswas, C., Li, X.-Q., Nemec, R. E., and Tool, B. P. (1989)The Biology of Hyaluronan, Ciba Foundation Symposium 143, pp. 150-169,Wiley, Chichester, England; Laurent, T. C., and Fraser, J. R. E. (1992)FASEB J. 6, 2397-2404; Kimata, K., Honma, Y., Okayama, M., Oguri, K.,Hozumi, M., and Suzuki, S. (1983) Cancer Res. 43, 1347-1354).

Biosysthesis of hyaluronan has been widely studied using a procaryote,Streptococci. A recent report revealed that a structural gene ofhyaluronan synthase derived from Streptococcus pyogenes which is aprocaryote was isolated (DeAngelis, P. L., Papaconstantinou, J., andWeigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). In contrast,little is known about the biosynthesis mechanism of hyaluronan ineucaryotes. Attempts have been made to purify eucaryotic hyaluronansynthase. However, some reports showed that the obtained enzyme lost itsactivity (Mian, N. (1986) Biochem. J. 237, 343-357; Ng, K. F., andSchwartz, N. B. (1989) J. Biol. Chem. 264, 11776-11783; Klewes, L.,Turley, E. A., and Prehm, P. (1993) Biochem. J. 290, 791-795). Any DNAencoding a polypeptide of eucaryotic hyaluronan synthase is not known.

If a polypeptide of hyaluronan synthase derived from eucaryotes,particularly human, and a DNA encoding it are obtained, they would beuseful for treatments, including gene therapy, of diseases caused bydecreased expression of hyaluronan in humans. In addition, thesesubstances would also be useful for gene therapy for suppressingmetastasis of cancer using an antisense DNA, RNA, or the like as well asdevelopment of hyaluronan synthase-specific inhibitors.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a polypeptide of ahyaluronan synthase of human origin and a DNA encoding the polypeptide.

The present inventors intensively investigated to achieve the aboveobject and, as a result, succeeded in cloning a cDNA encoding apolypeptide of hyaluronan synthase, which has hyaluronan synthaseactivity, from cells of an organism except human and, by using afragment derived from the cDNA, cloning a cDNA encoding a polypeptide ofa hyaluronan synthase of human origin. Thus, the present invention wascompleted.

The present invention provides a DNA encoding the following polypeptide(a) or (b):

(a) a polypeptide of a hyaluronan synthase of human origin; and

(b) a partial polypeptide of the polypeptide (a).

Further, the present invention also provides a DNA encoding any one ofthe following polypeptides (a) to (c):

(a) a polypeptide having the amino acid sequence shown by SEQ ID NO: 4;

(b) a polypeptide having the amino acid sequence shown by SEQ ID NO: 4,which has a substitution, deletion or insertion of one or more aminoacid residues that does not substantially lower an activity ofsynthesizing hyaluronan; and

(c) a partial polypeptide of the polypeptide (a) or (b).

The above-described DNA preferably encodes the whole or the amino acidsequence shown by SEQ ID NO: 4. It also preferably has at least a partof the nucleotide sequence shown by SEQ ID NO: 1, more preferably has anucleotide sequence of from position 149 to position 1777 of thenucleotide sequence shown by SEQ ID NO: 1. In the present invention, theDNA of the present invention includes a DNA or an RNA complementary tothe DNA.

Furthermore, the present invention provides a polypeptide of ahyaluronan synthase of human origin or a part thereof (hereinafterreferred to "the polypeptide of the present invention") encoded by theabove-described DNA of the present invention.

The term "part of the polypeptide" used herein means a part having anactivity or function such as hyaluronan synthase activity andimmunogenicity, or a part, the nucleotide sequence of which is specificto the hyaluronan synthase and can be used as a primer or a probe.

Further, the term "at least part of the nucleotide sequence" used hereinmeans a part encoding a part of the polypeptide having an activity orfunction such as hyaluronan synthase activity and immunogenicity, or thepart which is specific to the hyaluronan synthase and can be used as aprimer or a probe.

The following explains the embodiments of the present invention indetail.

I. THE DNA OF THE PRESENT INVENTION

The DNA of the present invention is the one encoding at least part of apolypeptide of a hyaluronan synthase of human origin, specifically oneencoding at least part of a polypeptide having the amino acid sequenceshown by SEQ ID NO: 4. The polypeptide of the hyaluronan synthase ofhuman origin or the polypeptide having the amino acid sequence shown bySEQ ID NO: 4, which is encoded by the DNA of the present invention, mayhave a substitution, deletion or insertion of one or more amino acidresidues that does not substantially lower the activity of synthesizinghyaluronan. The DNA of the present invention is preferably a DNAencoding at least part of the polypeptide of a hyaluronan synthase ofhuman origin, and encoding the whole or a part of the amino acidsequence shown by SEQ ID NO: 4, or a DNA encoding at least part of thepolypeptide of the hyaluronan synthase of human origin, which has theamino acid sequence shown by SEQ ID NO: 4, in which a substitution,deletion or insertion of one or more amino acid residues that does notsubstantially lower the activity of synthesizing hyaluronan may bepresent. More preferably, the DNA of the present invention is a DNAencoding the whole of the amino acid sequence shown by SEQ ID NO: 4. TheDNA of the present invention is still more preferably a DNA having atleast part of the nucleotide sequence shown by SEQ ID NO: 1. A specificexample of the DNA is one having the nucleotide sequence of fromposition 149 to position 1777 of the nucleotide sequence shown by SEQ IDNO: 1. The DNA of the present invention may have a substitution,deletion, or insertion of one or more nucleotides as long as it hassubstantially the same nucleotide sequence as shown by SEQ ID NO: 1 anddoes not substantially lower the hyaluronan-synthesizing activity of thepolypeptide of hyaluronan synthase encoded by the above nucleotidesequence. In other words, the DNA of the present invention includes DNAencoding a polypeptide of hyaluronan synthase having the above-describedsubstitution, deletion, or insertion of nucleotide(s).

A specific example of the DNA of the present invention is a DNA havingthe nucleotide sequence of from position 149 to position 1777 of thenucleotide sequence shown by SEQ ID NO: 1. One of ordinary skill in theart would readily understand that the DNA of the present inventioninclude DNAs having the nucleotide sequences different from that asdescribed above due to degeneracy of the genetic codes.

Also, the DNA of the present invention may be either a coding singlestrand encoding only the polypeptide of hyaluronan synthase or adouble-stranded chain composed of the above single strand and a DNAstrand having complementary sequence thereto.

Since the nucleotide sequence of the DNA of the present invention wasrevealed by the present invention, the DNA can be synthesized based onthe sequence. The DNA is also obtained by amplifying the DNA of thepresent invention from human chromosomal DNA or mRNA by polymerase chainreaction (PCR) method using oligonucleotide primers prepared based onthe sequence. The DNA of the present invention was obtained for thefirst time by the cDNA cloning comprising the following steps as alsodescribed in the example below.

(1) Cloning of a cDNA Encoding a Polypeptide of a Hyaluronan Synthase ofMouse Origin:

i) selection of mouse cells capable of high level production ofhyaluronan and preparation of mouse mutant cells deficient inhyaluronan-synthesizing ability;

ii) isolation of a poly(A)⁺ RNA containing an mRNA corresponding to thepolypeptide of hyaluronan synthase;

iii) construction of a mouse cDNA library;

iv) introduction of the cDNA library constructed in iii) into the mutantcells deficient in hyaluronan-synthesizing ability prepared in i)(transfection);

v) selection of cells capable of synthesizing hyaluronan from the cellstransformed in iv);

vi) Recovery of a plasmid DNA from the cells selected in

v), transformation of Escherichia coli cells with the plasmid DNA, andrecovery of the plasmid DNA from the E. coli cells; and

vii) isolation of a cDNA encoding the polypeptide of the hyaluronansynthase of mouse origin by repeating three more times the above stepsiv) to vi) using the recovered plasmid DNA.

(2) Cloning of a cDNA Encoding a Polypeptide of the Hyaluronan Synthaseof Human Origin:

i) preparation of a probe for screening a human cDNA library based onthe result of sequencing of the cDNA isolated in vii) of (1) above;

ii) screening of cDNA clones of human hyaluronan synthase using theprobe prepared in i); and

iii) nucleotide sequencing.

The method of producing the DNA of the present invention is not to belimited to the above method. The DNA of the present invention can beprepared by PCR method as described above or other known cDNA cloningmethods.

An example of the method of producing the DNA of the present inventionis described in detail below.

1. Cloning of a cDNA Encoding a Polypeptide of Hyaluronan SynthaseDerived from an Organism Except Human

(1) Selection of Cell Lines Capable of High Level Production ofHyaluronan and Preparation of Mutant Cells Deficient inHyaluronan-synthesizing Ability

(1-1) Selection of Cells Capable of High Level Production of Hyaluronan

Cells capable of high level production of hyaluronan (hereinafter alsosimply referred to as "high level hyaluronan-producing cells") areselected using cells of an organism except human having the hyaluronansynthase activity. Though the cells of an organism except human havingthe hyaluronan synthase activity are not particularly limited,eucaryotic cells are preferable, mammalian cells are more preferable,and mouse cells are particularly preferable. Established cultured celllines (hereinafter also simply referred to as "cell line") arepreferably used in view of availability, easiness to handle, and abilityto proliferate. Mouse-derived cell lines are more preferable. Among themouse-derived cell lines, FM3A, a mouse mammary carcinoma cell line(Health Science Research Resources Bank; cell number, JCRB0701, or RIKENCell Bank; cell number, RCB0086) is particularly preferable.Furthermore, among FM3A, FM3A P15A that is a cell line selected takinghigh metastatic ability to lung as an index (Honma, Y., Kasukabe, T.,and Hozumi, M. (1981) Gann 72, 898-905; Kimata, K., Honma, Y., Okayama,M., Oguri, K., Hozumi, M., and Suzuki, S. (1983) Cancer Res. 43,1347-1354) is most preferable because of its high hyaluronan synthaseactivity.

Culture media used for culturing the above-described cultured cells arenot particularly limited as long as cells can grow therein. Any mediaknown in the field of cell culture can be used. For example, Eagle'sminimum essential medium or the like is preferable since it has beenwidely used in usual culturing, readily available, and allows theabove-described cells to grow. The pH of the medium is adjustedpreferably to the neutral region, particularly to pH 7.0.Heat-inactivated bovine serum is preferably added to the medium to about10%. Furthermore, amino acids and vitamins are preferably added to themedium in the order of double with respect to the usual amounts.Substances such as penicillin and streptomycin can be added to preventproliferation of contaminating microorganisms. The above-described cellscan be maintained or proliferated with the above medium in a dish or aroller bottle by the usual culturing method. The culture can bepreferably performed in a carbon dioxide incubator; the concentrationsof carbon dioxide and air in the incubator are preferably adjusted to 3to 7% and 97 to 93%, respectively. The temperature is preferablyadjusted to about 37 to 38° C.

The method of selecting the high level hyaluronan-producing cells is notparticularly limited as long as it enables evaluation of thehyaluronan-producing ability of the cells. For example, a methodutilizing fixed erythrocyte exclusion assay (Knudson, W. and Knudson, C.B. (1991) J. Cell. Sci. 99, 227-235) can be used. In the fixederythrocyte exclusion assay, the degree of formation of extracellularhyaluronan matrix can be observed taking as an index the distance thatfixed erythrocytes cannot come close to the cells. More specifically,the longer the distance that fixed erythrocytes cannot come close to thecells is, the thicker the hyaluronan matrix is formed. Thehyaluronan-producing ability of the cells can be evaluated by observingthe degree of formation of extracellular hyaluronan matrix using thefixed erythrocyte exclusion assay. The example as described belowspecifically demonstrates the fixed erythrocyte exclusion assay.

The high level hyaluronan-producing cells as selected in the abovemethod are used in the preparation of a poly(A)⁺ RNA.

(1-2) Preparation of Mutant Cells Deficient in theHyaluronan-synthesizing Ability

Mutant cells deficient in the hyaluronan-synthesizing ability(hereinafter also simply referred to as "cells incapable of synthesizinghyaluronan") can be obtained by mutagenizing the high levelhyaluronan-producing cells as selected in (1-1) above by exposing thecells to physical or chemical stimulation to some extent that the cellsdo not die. The physical stimulation includes, for example, treatmentwith radiation such as X-ray or γ-ray. The chemical stimulation includestreatment with a mutagen such as an alkylating agent. Preferablealkylating agents include nitrosoguanidine and its derivatives, forexample, N-methyl-N'-nitro-N-nitrosoguanidine or the like. WhenN-methyl-N'-nitro-N-nitrosoguanidine is used as a mutagen, itsconcentration is preferably about 0.5 μg/ml. The cells incapable ofsynthesizing hyaluronan can be selected and recovered from the cellsmutagenized by the above method, according to a method for evaluatingthe hyaluronan-producing ability, such as the fixed erythrocyteexclusion assay (described in detail in the following example).

(1-3) Preparation of Cells Incapable of Synthesizing Hyaluronan Used asa Host into which a Recombinant DNA is Transfected

When the cells incapable of synthesizing hyaluronan prepared in (1-2)above are used as a host into which a recombinant DNA is transfected asin (4) shown below, the cells have been preferably transformed so as toexpress polyoma large T antigen. For the transformation, the cells havebeen preferably transformed with a plasmid carrying the polyoma Tantigen gene. An example of the plasmid carrying the polyoma T antigengene is pdl3027 plasmid (Nagata, Y., Yamashiro, S. Yodoi, J., Lloyd, K.O, Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089).The thus-obtained cells incapable of synthesizing hyaluronan andexpressing polyoma large T antigen (hereinafter also simply referred toas "T antigen-expressing cells incapable of synthesizing hyaluronan")can be used in (4) below as a host into which a recombinant DNA istransfected.

(2) Isolation of a Poly(A)⁺ RNA Containing an mRNA Corresponding to thePolypeptide of Hyaluronan Synthase from the High LevelHyaluronan-producing Cells Prepared in (1-1) Above

(2-1) Preparation of the Total RNA

The total RNA can be obtained by the known method (Kingston, R. E.(1991) Current Protocols in Molecular Biology, Suppl. 14, Unit 4.2,Greene Publishing Associates and Wiley Interscience, New York, etc.).Although a total RNA can be obtained from the high levelhyaluronan-producing cells prepared in (1-1) above by the method usuallyused for preparing a total RNA, a preferable method includes theguanidine thiocyanate/CsCl method (Kingston, R. E. (1991) CurrentProtocols in Molecular Biology, Suppl. 14, Unit 4.2, Greene PublishingAssociates and Wiley Interscience, New York).

(2-2) Preparation of a Poly(A)⁺ RNA

A poly(A)⁺ RNA can be purified from the total RNA obtained in (2-1)above by oligo-(dT) cellulose column chromatography or the like.

(3) Preparation of a cDNA Library

(3-1) Synthesis of cDNA

cDNA can be synthesized by the reverse transcriptase reaction using thepoly(A)⁻ RNA prepared in (2) above as a template. Specifically, themethods usually used in the field of gene engineering may be used.Alternatively, a commercially available cDNA synthesizing kit may beused. Though oligo(dT), which is usually used as a primer, can be usedas a primer in the reverse transcriptase reaction, a random oligonucleotide primer is preferably used.

(3-2) Preparation of a cDNA Library

A cDNA library can be obtained by ligating the cDNA obtained in (3-1)above to a cloning vector.

Although the cloning vector to which the cDNA is ligated is notparticularly limited, an expression vector functioning in mammaliancells is preferably used. In order to make the subsequent procedurereadily operable, it is preferable to use a shuttle vector having areplication origin functioning in cells such as Escherichia coli (E.coli). A preferable vector may be pcDNAI (manufactured by InvitrogenCo.). The cDNA obtained in (3-1) above is ligated to the above-describedcloning vector to give a recombinant DNA (cDNA library).

(4) Transfection of the Recombinant DNA Prepared in (3) Above into theCells Incapable of Synthesizing Hyaluronan

The recombinant DNA (cDNA library) obtained by ligating the cDNA to thecloning vector is transfected into host cells. The host cells to be usedshould be selected depending on the cloning vector used. For example,when an expression vector functioning in mammalian cells is used as acloning vector, host cells should be mammalian cells. Alternatively, thecDNA library may be prepared by selecting host cells first and thenselecting a cloning vector suitable for the host cells.

When cloning is performed using the presence of hyaluronan-synthesizingability as an index, it is preferable to use as host cells the cellsincapable of synthesizing hyaluronan as prepared in (1) above. The Tantigen-expressing cells incapable of synthesizing hyaluronan preparedin (1-3) above are most preferably used.

The DNA can be transfected into the host cells by the method usuallyused in the field of gene engineering. A commercially available reagentfor transformation can also be used. As such a reagent, Lipofectoamine™reagent (Life Technologies, Inc) is suitably used. The transformed Tantigen-expressing cells incapable of synthesizing hyaluronan arecultured using the medium and the method as described in (1-1) above.Though the culturing period is not particularly limited, about 64 hoursare preferable.

(5) Selection of Cells Capable of Synthesizing Hyaluronan fromTransformants Obtained in (4) Above

(5-1) Preparation of Labeled Hyaluronan-binding Protein Used forDetecting Hyaluronan

Among the host cells into which the cDNA library has been transfected,the T antigen-expressing cells incapable of synthesizing hyaluronan,into which the recombinant DNA containing the cDNA encoding thepolypeptide of hyaluronan synthase has been transfected, express thepolypeptide of hyaluronan synthase by culturing the cells by the methodas described in (4) above so that hyaluronan can be synthesized andaccumulated extracellularly. The T antigen-expressing cells incapable ofsynthesizing hyaluronan, into which the recombinant DNA containing cDNAencoding the polypeptide of hyaluronan synthase has been transfected,can be selected by detecting cells in which hyaluronan is accumulatedextracellularly. Though the method of detecting extracellularlyaccumulated hyaluronan is not particularly limited, the detection ispreferably performed by using a protein capable of binding to hyaluronan(herein also referred to as "hyaluronan-binding protein") that islabeled (herein also referred to as "labeled hyaluronan-bindingprotein"). The hyaluronan-binding protein is not particularly limited.Examples thereof include aggrecan, neurocan, versican, link protein,hyaluronectin, hyaluronan-binding protein derived from human synovialfluid, brain hyaluronan-binding protein, and CD44. Among these, aggrecanis preferably used. Particularly, the hyaluronan-binding region ofaggrecan (herein also abbreviated as "HABR") is preferably used.

A substance used to label the hyaluronan-binding protein is notparticularly limited. Examples thereof include biotin, avidin (includingstreptoavidin), fluorescent substances, enzymes, and radioisotopes.Biotin is preferably used. The method of labeling the hyaluronan-bindingprotein using the above labels is not particularly limited. A knownlabeling method can be used.

When biotin is used to label the hyaluronan-binding protein, forexample, avidin labeled with a fluorescent substance is used to detectthe biotin. A commercially available labeled avidin can also be used.Further, biotinated HABR, which is an example of the labeledhyaluronan-binding protein, will be described in the following examplefor its specific preparation method.

(5-2) Selection of Cells Capable of Synthesizing Hyaluronan

The T antigen-expressing cells incapable of synthesizing hyaluronantransformed in (4) above are contacted with the labeledhyaluronan-binding protein obtained in (5-1) above and the mixture isincubated. The cells capable of synthesizing hyaluronan can be selectedby detecting and selecting cells in which hyaluronan is accumulatedextracellularly. Further, when the hyaluronan-binding protein is labeledwith biotin, the cells need to be incubated with labeled avidin tocontact together after incubation with the biotin-labeledhyaluronan-binding protein. The cells are preferably washed after everyincubation. The incubation conditions are not particularly limited aslong as binding of hyaluronan to the hyaluronan-binding protein is notinhibited. Preferably, the incubation is performed at a neutral pH andon ice (0° C.). Though the incubation period is not also particularlylimited, it is preferably about 1 hour.

After the incubation, a known detection method for the label is suitablyselected depending upon the type of the label used, thereby detectingcells (positively stained cells) on which the label is foundextracellularly (on the cell surface). Thus, the cells capable ofsynthesizing hyaluronan can be selected. When a fluorescent substance isused as a label, detection of the label and selection of the cells canbe concurrently done by means of flow cytometry.

(6) Recovery of a Plasmid DNA from the Cells Selected in (5) Above,Transformation of E. coli with the Recovered Plasmid DNA, and Recoveryof the Plasmid DNA from the Transformed E. coli

A plasmid DNA (a recombinant DNA) is recovered from the cells capable ofsynthesizing hyaluronan selected in (5) above and the resultingrecombinant DNA is used to transform Escherichia coli (E. coli). Themethod of recovering plasmid DNA is performed with the commonly usedtechnique in the field of gene engineering. It is not particularlylimited thereto, but the method of Hirt (Hirt, B. (1967) J. Mol. Biol.26, 365-369) is preferably used.

E. coli is transformed with the recovered DNA and cultured by the usualmethod for culturing E. coli. The plasmid DNA (recombinant DNA) is thenrecovered from the resulting E. coli.

(7) Isolation of the cDNA

As described above, the cDNA encoding the polypeptide of hyaluronansynthase can be isolated. In order to confirm that the cDNA obtainedactually encodes the polypeptide of hyaluronan synthase, the above steps(4) to (6) are preferably repeated about three more times.

The cDNA thus obtained is subjected to sequencing as it is or aftersubcloned into an appropriate plasmid. 2. Cloning of the cDNA encodingthe polypeptide of human hyaluronan synthase

(1) Preparation of Probes for Screening of the cDNA Library

The nucleotide sequence of the cDNA corresponding to the polypeptide ofhyaluronan synthase derived from an organism except human determined asdescribed above is compared with that of the cDNA of hyaluronan synthasederived from a microorganism, preferably Streptococcus to determine theregions that are conserved (herein also simply referred to as "conservedregions"), which can be used as a 5' primer and a 3' primer inpolymerase chain reaction (PCR). Thereafter, each conserved region issynthesized.

Labeled DNA probes used for screening the cDNA library can be preparedby the DNA labeling method usually used in the field of gene engineeringusing the primers prepared based on the conserved regions (the 5' primerand the 3' primer) from the hyaluronan synthase gene derived from anorganism except human as obtained in the item 1 above. In presentinvention, a radioisotope and a fluorescent substance can be used as alabel. A fluorescent substance is preferably used. The labeled DNA probecan be efficiently prepared in a large quantity by PCR.

(2) Screening of Clones of cDNA of Human Hyaluronan Synthase Using theProbes Prepared in (1) Above

(2-1) Preparation of a cDNA Library

A total RNA is prepared from cells derived from human and a poly(A)⁺ RNAis prepared from the total RNA. A cDNA can be synthesized by the reversetranscriptase reaction using the poly(A)⁺ RNA as a template. Theseprocedures are all performed by the methods usually used in the field ofgene engineering. Specifically, the methods as described in (2) and (3)in item 1 above may be used.

The cDNA is ligated to a cloning vector. Though the cloning vector isnot particularly limited, for example, λgt11 digested with EcoRI ispreferably used. Alternatively, a commercially available humancDNA-ligated cloning vector may be used.

During the process for achieving the present invention, cDNA clones ofhuman hyaluronan synthase were selected, as demonstrated in the example,from a human cDNA library using probes prepared based on the mouse cDNAsequence. The cDNA can be ligated to an expression vector functioning inmammalian cells and selected using the expression product as an index.

(2-2) Screening of the cDNA Clones of Human Hyaluronan Synthase

From the cDNA library obtained as described above, phage clones havingthe full-length of the cDNA of hyaluronan synthase can be selected byhybridization using the probe prepared in (1) above. The hybridizationcan be performed by the method usually used in the field of geneengineering, for example, plaque hybridization or the like. Plaques thathybridize with the probe can be isolated and identified by detecting thelabel bound to the probe. One of ordinary skill in the art canappropriately detect the label depending on the label used.

(3) Sequencing

A phage DNA is prepared from positive λgt11 clones selected in (2) aboveand digested with an appropriate restriction enzyme to cleave out thecDNA of hyaluronan synthase. The enzyme used for cloning the cDNA isused as the restriction enzyme. For example, when λgt11 digested withEcoRI is used as the vector, EcoRI is preferably used. The thus-obtainedcDNA is subjected to sequencing as it is or after subcloned into anappropriate plasmid. When the cDNA is subcloned, pcDNA3 plasmid vector(manufactured by Invitrogen Co.) is preferably used.

The DNA as obtained above may have a substitution, deletion, orinsertion of nucleotide(s) or the combination thereof, which result insubstitution, deletion, or insertion of one or two or more amino acidresidues of the polypeptide of hyaluronan synthase encoded by the DNA aslong as the hyaluronan-synthesizing activity of the polypeptide is notsubstantially lowered. The substitution, deletion, or insertion of thenucleotides can be introduced in the DNA sequence by synthesizing asequence having restriction enzyme cleavage sites at the both ends andcontaining the both sides of the mutated site and replacing it with thecorresponding non-mutated DNA sequence. The site-directed mutationmethod (Kramer, W. and Frits, H. J. (1987) Meth. in Enzymol. 154, 350;Kunkel, T. A. et al. (1987) Meth. in Enzymol. 154, 367) or the like canalso be used for introducing the substitution, deletion, or insertion,or the combination thereof into the DNA sequence.

The method of measuring the hyaluronan-synthesizing activity is wellknown in the art. Thus, one of ordinary skill in the art would readilydetermine the substitution, deletion, or insertion, or the combinationthereof that does not substantially lower the hyaluronan-synthesizingactivity.

II. THE POLYPEPTIDE OF THE PRESENT INVENTION

The polypeptide of the present invention is a polypeptide of thehyaluronan synthase of human origin or a part thereof, which is encodedby the DNA of the present invention. It is preferably a polypeptide ofthe hyaluronan synthase having the amino acid sequence (amino acidnumbers 1 to 543) shown by SEQ ID NO: 4.

Since the amino acid sequence is revealed by the present invention, thepolypeptide of the present invention can be synthesized based on theamino acid sequence. It is possible and preferable, however, to obtainthe polypeptide by expressing the DNA of the present invention.

For example, the DNA of the present invention is ligated to anappropriate expression vector functioning in mammalian cells and cellscarrying this vector are cultured in an appropriate medium to produceand accumulate the polypeptide of hyaluronan synthase in the culture (inthe cells and/or the medium). The polypeptide of hyaluronan synthase isthen extracted from the culture. In this way, the polypeptide ofhyaluronan synthase can be produced. In this procedure, the usually usedmethods for extracting and purifying enzymes can be used.

Specifically, examples of the extraction method include cell disruptiondue to, for example, ultrasonication, homogenization, osmotic shockprocedure, freezing and thawing method, treatment with a surfactant, andthe combined process thereof. Further, specific examples of thepurification methods include salting out with ammonium sulfate or sodiumsulfate, centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,reverse phase chromatography, gel filtration, gel permeationchromatography, affinity chromatography, electrophoresis, and thecombined process thereof.

The DNA of the present invention can be expressed using a host-vectorsystem usually used for producing proteins. The host-vector system ispreferably an animal cell system, particularly, a mammalian cell system.The DNA of the present invention may be expressed alone or as a fusionprotein together with another protein. The full-length of the DNA may beexpressed. Alternatively, part of the DNA may be expressed to produce apartial peptide.

The polypeptide of hyaluronan synthase as produced above or its partialpeptide, or a fusion protein thereof with another protein can be used toproduce antibodies that bind to the hyaluronan synthase. The antibodiescan be prepared by the usual methods for producing antibodies.Monoclonal antibodies that bind to the hyaluronan synthase can also beproduced by the usual methods.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following example.

First, the methods commonly used throughout the example are described.

1. Fixed Erythrocyte Exclusion Assay

Cells were inoculated on a 35-mm tissue culture dish and cultured at 37°C. for 3 days. The culture medium was replaced with 750 μl of asuspension of fixed sheep erythrocytes (manufactured by Inter-CellTechnologies, Inc.) (10⁸ cells/ml). After the dish was allowed to standfor 10 minutes, extracellular hyaluronan matrix were observed as theregion that the erythrocytes could not come close to. The observationwas performed using a phase-contrast microscope (OLYMPUS IMT-2;manufactured by Olympus Optical Co.) with 200-fold magnification.

2. Preparation of Biotinated Hyaluronan-binding Region (b-HABR)

Aggrecan was extracted from bovine nasal cartilage with a guanidinesolution and purified by cesium chloride density gradient centrifugationunder association and dissociation conditions. The hyaluronan-bindingregion of the purified aggrecan (2 mg) was protected by hyaluronan andthe protected aggrecan was reacted with 0.57 mg of NHS-LC-biotin(manufactured by Pierce) at room temperature for 2 hours forbiotination. The biotinated aggrecan was partially digested with trypsinand the digested product was subjected to gel filtration using aSephacryl S-300 column (manufactured by Pharmacia) and then hyaluronanaffinity chromatography (Tengblad, A. (1979) Biochem. Biophys. Acta 578,281-289). Thus, b-HABR was purified.

A production example of the DNA of the present invention is describedbelow.

1. Cloning of a cDNA Encoding a Polypeptide of a Hyaluronan Synthase ofMouse Origin

(1) Selection of High Level Hyaluronan-producing Cells and Preparationof Cells Incapable of Synthesizing Hyaluronan

(1-1) Selection of High Level Hyaluronan-producing Cells

FM3A P-15A having high metastatic ability to lung was selected andestablished as a cell line derived from mouse mammary carcinoma cellline FM3A (Health Science Research Resources Bank; cell number,JCRB0701), which is one of mouse-derived established cell lines. FM3AP-15A cells were cultured in Eagle's minimum essential medium(manufactured by Nissui Pharmaceutical) containing 10% heat-inactivatedbovine serum, doubled concentrations of amino acids and vitamins,penicillin, and streptomycin in a 100-mm petri dish (manufactured byFalcon. No. 1005). Culture was performed under the condition of 37° C.in 5% CO₂.

The degree of formation of extracellular hyaluronan matrix in the FM3AP-15A culture was examined by the fixed erythrocyte exclusion assay.Cells that extracellularly formed hyaluronan matrix in a larger amountcompared with the other cells were collected and pooled. The resultingcells (high level hyaluronan-producing cells) were designated as FM3AHA1.

(1-2) Preparation of Cells Incapable of Synthesizing Hyaluronan

The high level hyaluronan-producing cells selected in (1-1) above weretreated with 0.5 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine(manufactured by Nacalai Tesque Co.) and examined for the degree offormation of extracellular hyaluronan matrix by fixed erythrocyteexclusion assay. Cells that did not extracellularly form hyaluronanmatrix were collected and pooled. The thus-obtained cells incapable ofsynthesizing hyaluronan were designated as HAS⁻ cells.

(1-3) Preparation of HAS⁻ Cells that Express Polyoma Large T Antigen

Plasmid pdl3027 containing the polyoma T antigen gene (given by Dr. K.Furukawa (Nagasaki University) and Dr. C. Basilico (New YorkUniversity)) and plasmid pSV2neo (manufactured by CLONTECH Co.) weretransfected into HAS⁻ cells as obtained above. The cells were selectedthrough treatment with 500 μg/ml G-418 (manufactured by Gibco Co.).Thus, HAS⁻ cells that constantly express polyoma large T antigen (hereinalso referred to as "HAS-P cells") were prepared.

(2) Isolation of a Poly(A)⁺ RNA from FM3A HA1

(2-1) Preparation of a Total RNA

A total RNA was prepared from FM3A HA1 by the guanidine thiocyanate/CsClmethod (Kingston, R. E. (1991) Current Protocols in Molecular Biology,Suppl. 14, Unit 4.2, Greene Publishing Associates and WileyInterscience, New York).

(2-2) Preparation of a Poly(A)⁺ RNA

A poly(A)⁺ RNA was purified from the total RNA obtained in (2-1) aboveby oligo-(dT) cellulose column chromatography.

(3) Preparation of a cDNA Library

(3-1) Synthesis of cDNA

cDNA was synthesized by reverse transcriptase reaction using thepoly(A)⁻ RNA obtained in (2-2) above as a template, and randomoligonucleotide primers as primers.

(3-2) Preparation of a cDNA Library

The cDNA obtained in (3-1) above was ligated to pcDNAI (manufactured byInvitrogen Co.), an expression vector functioning in mammalian cells, toprepare a cDNA library.

(4) Transformation by the cDNA Library

The cDNA library prepared in (3-2) above was transfected into HAS-Pcells prepared in (1-3) above using Lipofectoamine™ reagent,manufactured by Life Technologies, Inc. The transformed HAS-P cells werecultured in the medium under the conditions as described in (1-1) abovefor 64 hours.

(5) Selection of Cells Capable of Synthesizing Hyaluronan fromTransformants

After 64-hour culturing as described in (4) above, the transformantswere washed with a cooled medium (Cosmedium001 (manufactured by CosmoBio)) and then suspended in the same medium but containing 50 μg/ml ofb-HABR. After 1 hour, the cells were washed with cooled Cosmedium001 andsuspended in the same medium but containing avidin labeled withfluorescein (fluorescein avidin DCS, manufactured by VectorLaboratories, Inc.). After 1 hour, the cells were washed with cooledphosphate-buffered saline (PBS) containing 5% fetal calf serum andsuspended in the same solution. Positively stained cells were selectedusing a flow cytometer (EPICS Elite Flow Cytometer, manufactured byCoulter Electronics, Inc.).

(6) Recovery of a Plasmid DNA from the Cells Selected in (5) Above,Transformation of E. coli with the Recovered Plasmid DNA, and Recoveryof the Plasmid DNA from E. coli Transformants

A plasmid DNA was recovered from the cells selected in (5) above by themethod of Hirt (Hirt, B. (1967) J. Mol. Biol. 26, 365-369).Specifically, this procedure was performed in the following manner. Thecells selected in (5) above were dissolved in 100 μl of 0.6% sodiumdodecyl sulfate (SDS)/10 mM ethylenediamine-tetraacetic acid (EDTA)solution and the mixture was allowed to stand at the room temperaturefor 20 minutes. Twenty-five ml of 5M NaCl was then added thereto and themixture was allowed to stand overnight on ice. The solution was treatedwith phenol and chloroform and then precipitated with ethanol to recovera plasmid DNA. The recovered plasmid DNA was used to transform E. coliMC1061/P3 (manufactured by Invitrogen Co.) by electroporation. Theresulting transformants were cultured and the plasmid DNA was thenrecovered therefrom.

(7) Isolation of the cDNA Encoding the Polypeptide of HyaluronanSynthase

The above steps (4) to (6) (steps of transformation and selection) wererepeated three more times using the plasmid DNA recovered from thetransformed E. coli MC1061/P3 obtained in (6) above to isolate cDNAencoding the polypeptide of hyaluronan synthase. The thus-isolated cDNAencoding the polypeptide of hyaluronan synthase was designated aspcDNAI-HAS.

(8) Determination and Analysis of the cDNA Sequence Encoding thePolypeptide of Hyaluronan Synthase

The cDNA clone isolated in (7) above was subcloned into a plasmidvector, pcDNA3 (manufactured by Invitrogen Co.). The thus-obtainedrecombinant plasmid was designated as pcDNA3-HAS. pcDNA3-HAS wasdenatured with alkali and its nucleotide sequence of both sense andantisense directions was determined using [³⁵ S]dCTP and deazaGTP kit(manufactured by U.S. Biochemical Co.) and DNA polymerase (Sequenase ver2.0, manufactured by U.S. Biochemical Co.).

DNA synthesis for sequencing was performed using primers synthesizedbased on T7, SP6, and the nucleotide sequence existing at about 250 bpintervals in pcDNA3. The thus-obtained DNA sequence was analyzed byGENETYX-MAC computer program (manufactured by Software Development Co.).The nucleotide sequence and the deduced amino acid sequence therefromwere analyzed utilizing a database for analyzing nucleic acids andproteins (EMBL-GDB, Release44, and NBRF-PDB, Release 45). The amino acidsequence of the polypeptide of hyaluronan synthase originating fromStreptococcus (Streptococcus pyogenes) was compared with that of thepolypeptide of the hyaluronan synthase originating from mouse. Tworegions that conserved in both of the polypeptides of hyaluronansynthase were found and designated as HAS-1 and HAS-2, respectively. Thenucleotide sequences corresponding to these regions are shown by SEQ IDNO: 2 (HAS-1) and SEQ ID NO: 3 (HAS-2).

2. Production of the DNA of the Present Invention

(1) Preparation of Probes for Screening a Human cDNA Library

An oligonucleotide having the sequence of HAS-1 and that having thesequence of HAS-2 were synthesized.

Fluorescence-labeled DNA probes used for screening the cDNA library wereprepared from the mouse-origin hyaluronan synthase gene obtained in theitem 1 above by PCR method with ECL™ probe-amp reagents (manufactured byAmersham Co.) using primers produced based on the conserved regions(HAS-1 and HAS-2; using as a 5' primer an oligonucleotide having thesequence of HAS-1 and as a 3' primer an oligonucleotide having thesequence of HAS-2). PCR amplification was performed 30 cycles usingGeneAmp PCR reagent kit (manufactured by Takara Shuzo) (Each cycleconsisted of denaturation at 95° C. for 1 minute, annealing at 50° C.for 2 minutes, and extension at 72° C. for 3 minutes.). The PCR productswere analyzed by 2% agarose gel electrophoresis.

(2) Screening of the cDNA Clones of Human Hyaluronan Synthase Using theProbes Prepared in (1)

For isolating a human homolog of the hyaluronan synthase gene, humanfetal brain cDNA library constructed in λgt11 (manufactured by CLONTECH)was mixed with indicator bacteria and melted soft agar. The mixture waslayered on an agar plate and cultured. As a result, 1×10⁶ plaques wereformed.

The plaques derived from the λgt11 cDNA library as obtained above werescreened. The plaques were transferred onto a commercially availablenylon membrane (Hybond N⁺ ™ nylon membrane, manufactured by Amersham)and the phage DNA was fixed on the nylon membrane by the alkalinefixation method recommended in the instructions appended to the product.

This filter was allowed to hybridize with the probes prepared in (1)above in 5×SSC containing 0.1% SDS, 5% (w/v) dextran sulfate, and 100μg/ml denatured salmon sperm DNA at 60° C. for 12 hours. The filter waswashed with 1×SSC containing 0.1% SDS at 60° C. for 15 minutes, then0.5×SSC containing 0.1% SDS at 60° C. for 15 minutes. Positive cloneswere detected with ECL™ detection kit (manufactured by Amersham).

(3) Sequencing

The recombinant vector was purified from the λgt11 positive clones anddigested with EcoRI to excise the cDNA insert as a single fragment. Thisfragment was subcloned into a plasmid vector, pcDNA3 (manufactured byInvitrogen Co.). The nucleotide sequence of the isolated cDNA wasdetermined by repeatedly sequencing both strands of the alkali-denaturedplasmid DNA using [³⁵ S]dGTP and deazaGTP kit and DNA polymerase(Sequenase version 2.0, manufactured by U.S. Biochemical Co.). DNAsynthesis for sequencing was started with primers synthesized based onT7, SP6, and the nucleotide sequence existing at about 250 intervals inpcDNA3. The DNA sequences obtained were compiled and analyzed usingGENETYX-MAC computer program (manufactured by Software Development Co.).The nucleotide sequence and the deduced amino acid sequence therefromwere analyzed using a database for analyzing nucleic acids and proteins(EMBL-GDB, Release44, and NBRF-PDB, Release45). The determinednucleotide sequence is shown by SEQ ID NO: 1. The amino acid sequenceencoded by the open reading frame existing in this nucleotide sequence(from position 149 to position 1777) is shown by SEQ ID NO: 4.

INDUSTRIAL APPLICABILITY

The polypeptide of the present invention can be used for mass productionof hyaluronan, which is used as an ingredient of medicines and cosmeticsat present, in an industrial scale, and for medicines for diseasescaused by decreased expression of hyaluronan. The polypeptide of thepresent invention can also be used for development of hyaluronansynthase-specific inhibitors. Furthermore, the polypeptide can be usedas an antigen to produce antibodies specific to the hyaluronan synthase.The DNA of the present invention can be used for mass production of thepolypeptide of hyaluronan synthase in an industrial scale, and for genetherapy for diseases caused by decreased expression of hyaluronan.Moreover, an antisense DNA and an antisense RNA of the DNA of presentinvention can be used for gene therapy for metastasis inhibitors.

The DNA of the present invention can also be used as a reagent forresearch purpose.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 4                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 2117                                                            <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (149)...(1777)                                                - <400> SEQUENCE: 1                                                           - gaattccggg cgcccgggac tcacgcccct tcctttcccc tctcgctccc ag - #caggacgc         60                                                                          - gcccaagccc actcctgcag cccgccgctg ctccggcctg gcccggaggg tg - #ctgaccat        120                                                                          #gcc gcc ggg       172c tggccctc atg acc tgg gcc tac                          #Ala Tyr Ala Ala Glyr Trp                                                     #           5  1                                                              - gtg ccg ctg gcc tcc gat cgc tac ggc ctc ct - #g gcc ttc ggc ctc tac          220                                                                          Val Pro Leu Ala Ser Asp Arg Tyr Gly Leu Le - #u Ala Phe Gly Leu Tyr           #     20                                                                      - ggg gcc ttc ctt tca gcg cac ctg gtg gcg ca - #g agc ctc ttc gcg tac          268                                                                          Gly Ala Phe Leu Ser Ala His Leu Val Ala Gl - #n Ser Leu Phe Ala Tyr           # 40                                                                          - ctg gag cac cgg cgg gtg gcg gcg gcg gcg cg - #g ggg ccg ctg gat gca          316                                                                          Leu Glu His Arg Arg Val Ala Ala Ala Ala Ar - #g Gly Pro Leu Asp Ala           #                 55                                                          - gcc acc gcg cgc agt gtg gcg ctg acc atc tc - #c gcc tac cag gag gac          364                                                                          Ala Thr Ala Arg Ser Val Ala Leu Thr Ile Se - #r Ala Tyr Gln Glu Asp           #             70                                                              - ccc gcg tac ctg cgc cag tgc ctg gcg tcc gc - #c cgc gcc ctg ctg tac          412                                                                          Pro Ala Tyr Leu Arg Gln Cys Leu Ala Ser Al - #a Arg Ala Leu Leu Tyr           #         85                                                                  - ccg cgc gcg cgc gtg cgc gtc ctc atg gtg gt - #g gat ggc aac cgc gcc          460                                                                          Pro Arg Ala Arg Val Arg Val Leu Met Val Va - #l Asp Gly Asn Arg Ala           #    100                                                                      - gag gac ctc tac atg gtc gac atg ttc cgc ga - #g gtc ttc gct gac gag          508                                                                          Glu Asp Leu Tyr Met Val Asp Met Phe Arg Gl - #u Val Phe Ala Asp Glu           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - gac ccc gcc acg tac gtg tgg gac ggc aac ta - #c cac cag ccc tgg gaa          556                                                                          Asp Pro Ala Thr Tyr Val Trp Asp Gly Asn Ty - #r His Gln Pro Trp Glu           #               135                                                           - ccc gcg gcg gcg ggc gcg gtg ggc gcc gga gc - #c tat cgg gag gtg gag          604                                                                          Pro Ala Ala Ala Gly Ala Val Gly Ala Gly Al - #a Tyr Arg Glu Val Glu           #           150                                                               - gcg gag gat cct ggg cgg ctg gca gtg gag gc - #g ctg gtg agg act cgc          652                                                                          Ala Glu Asp Pro Gly Arg Leu Ala Val Glu Al - #a Leu Val Arg Thr Arg           #       165                                                                   - agg tgc gtg tgc gtg gcg cag cgc tgg ggc gg - #c aag cgc gag gtc atg          700                                                                          Arg Cys Val Cys Val Ala Gln Arg Trp Gly Gl - #y Lys Arg Glu Val Met           #   180                                                                       - tac aca gcc ttc aag gcg ctc gga gat tcg gt - #g gac tac gtg cag gtc          748                                                                          Tyr Thr Ala Phe Lys Ala Leu Gly Asp Ser Va - #l Asp Tyr Val Gln Val           185                 1 - #90                 1 - #95                 2 -       #00                                                                           - tgt gac tcg gac aca agg ttg gac ccc atg gc - #a ctg ctg gag ctc gtg          796                                                                          Cys Asp Ser Asp Thr Arg Leu Asp Pro Met Al - #a Leu Leu Glu Leu Val           #               215                                                           - cgg gta ctg gac gag gac ccc cgg gta ggg gc - #t gtt ggt ggg gat gtg          844                                                                          Arg Val Leu Asp Glu Asp Pro Arg Val Gly Al - #a Val Gly Gly Asp Val           #           230                                                               - cgg atc ctt aac cct ctg gac tcc tgg gtc ag - #c ttc cta agc agc ctg          892                                                                          Arg Ile Leu Asn Pro Leu Asp Ser Trp Val Se - #r Phe Leu Ser Ser Leu           #       245                                                                   - cga tac tgg gta gcc ttc aat gtg gag cgg gc - #t tgt cag agc tac ttc          940                                                                          Arg Tyr Trp Val Ala Phe Asn Val Glu Arg Al - #a Cys Gln Ser Tyr Phe           #   260                                                                       - cac tgt gta tcc tgc atc agc ggt cct cta gg - #c cta tat agg aat aac          988                                                                          His Cys Val Ser Cys Ile Ser Gly Pro Leu Gl - #y Leu Tyr Arg Asn Asn           265                 2 - #70                 2 - #75                 2 -       #80                                                                           - ctc ttg cag cag ttt ctt gag gcc tgg tac aa - #c cag aag ttc ctg ggt         1036                                                                          Leu Leu Gln Gln Phe Leu Glu Ala Trp Tyr As - #n Gln Lys Phe Leu Gly           #               295                                                           - acc cac tgt act ttt ggg gat gac cgg cac ct - #c acc aac cgc atg ctc         1084                                                                          Thr His Cys Thr Phe Gly Asp Asp Arg His Le - #u Thr Asn Arg Met Leu           #           310                                                               - agc atg ggt tat gct acc aag tac acc tcc ag - #g tcc cgc tgc tac tca         1132                                                                          Ser Met Gly Tyr Ala Thr Lys Tyr Thr Ser Ar - #g Ser Arg Cys Tyr Ser           #       325                                                                   - gag acg ccc tcg tcc ttc ctg cgg tgg ctg ag - #c cag cag aca cgc tgg         1180                                                                          Glu Thr Pro Ser Ser Phe Leu Arg Trp Leu Se - #r Gln Gln Thr Arg Trp           #   340                                                                       - tcc aag tcg tac ttc cgt gag tgg ctg tac aa - #c gcg ctc tgg tgg cac         1228                                                                          Ser Lys Ser Tyr Phe Arg Glu Trp Leu Tyr As - #n Ala Leu Trp Trp His           345                 3 - #50                 3 - #55                 3 -       #60                                                                           - cgg cac cat gcg tgg atg acc tac gag gcg gt - #g gtc tcc ggc ctg ttc         1276                                                                          Arg His His Ala Trp Met Thr Tyr Glu Ala Va - #l Val Ser Gly Leu Phe           #               375                                                           - ccc ttc ttc gtg gcg gcc act gtg ctg cgt ct - #g ttc tac gcg ggc cgc         1324                                                                          Pro Phe Phe Val Ala Ala Thr Val Leu Arg Le - #u Phe Tyr Ala Gly Arg           #           390                                                               - cct tgg gcg ctg ctg tgg gtg ctg ctg tgc gt - #g cag ggc gtg gca ctg         1372                                                                          Pro Trp Ala Leu Leu Trp Val Leu Leu Cys Va - #l Gln Gly Val Ala Leu           #       405                                                                   - gcc aag gcg gcc ttc gcg gcc tgg ctg cgg gg - #c tgc ctg cgc atg gtg         1420                                                                          Ala Lys Ala Ala Phe Ala Ala Trp Leu Arg Gl - #y Cys Leu Arg Met Val           #   420                                                                       - ctt ctg tcg ctc tac gcg ccc ctc tac atg tg - #t ggc ctc ctg cct gcc         1468                                                                          Leu Leu Ser Leu Tyr Ala Pro Leu Tyr Met Cy - #s Gly Leu Leu Pro Ala           425                 4 - #30                 4 - #35                 4 -       #40                                                                           - aag ttc ctg gcg cta gtc acc atg aac cag ag - #t ggc tgg ggc acc tcg         1516                                                                          Lys Phe Leu Ala Leu Val Thr Met Asn Gln Se - #r Gly Trp Gly Thr Ser           #               455                                                           - ggc cgg cgg aag ctg gcc gct aac tac gtc cc - #t ctg ctg ccc ctg gcg         1564                                                                          Gly Arg Arg Lys Leu Ala Ala Asn Tyr Val Pr - #o Leu Leu Pro Leu Ala           #           470                                                               - ctc tgg gcg ctg ctg ctg ctt ggg ggc ctg gt - #c cgc agc gta gca cac         1612                                                                          Leu Trp Ala Leu Leu Leu Leu Gly Gly Leu Va - #l Arg Ser Val Ala His           #       485                                                                   - gag gcc agg gcc gac tgg agc ggc cct tcc cg - #c gca gcc gag gcc tac         1660                                                                          Glu Ala Arg Ala Asp Trp Ser Gly Pro Ser Ar - #g Ala Ala Glu Ala Tyr           #   500                                                                       - cac ttg gcc gcg ggg gcc ggc gcc tac gtg gg - #c tac tgg gtg gcc atg         1708                                                                          His Leu Ala Ala Gly Ala Gly Ala Tyr Val Gl - #y Tyr Trp Val Ala Met           505                 5 - #10                 5 - #15                 5 -       #20                                                                           - ttg acg ctg tac tgg gtg ggc gtg cgg agg ct - #t tgc cgg cgg cgg acc         1756                                                                          Leu Thr Leu Tyr Trp Val Gly Val Arg Arg Le - #u Cys Arg Arg Arg Thr           #               535                                                           - ggg ggc tac cgc gtc cag gtg tgagtccagc cacgcggat - #g ccgcctcaag            1807                                                                          Gly Gly Tyr Arg Val Gln Val                                                               540                                                               - ggtcttcagg ggaggccaga ggagagctgc tgggccccga gccacgaact tg - #ctgggtgg       1867                                                                          - ttctctgggc ctcagtttcc ctcctctgcc aaacgagggg gtcagcccaa ga - #ttcttcag       1927                                                                          - tctggactat attgggactg ggacttctgg gtctccaggg agggtattta tt - #ggtcagga       1987                                                                          - tgtgggattt gaggagtgga ggggaagggg tcctgctttc tcctcgttct ta - #tttaatct       2047                                                                          - ccatttctac tgtgtgatca ggatgtaata aagaatttta tttattttca aa - #aaaaaaaa       2107                                                                          #      2117                                                                   - <210> SEQ ID NO 2                                                           <211> LENGTH: 32                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: HAS-1, a region of the - # hyaluronan synthase       gene                                                                                which is conserved between S. pyo - #genes and mouse.                   - <400> SEQUENCE: 2                                                           #          32      aggt catgtacaca gc                                         - <210> SEQ ID NO 3                                                           <211> LENGTH: 35                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: HAS-2, a region of the - # hyaluronan synthase       gene                                                                                which is conserved between S. pyo - #genes and mouse.                   - <400> SEQUENCE: 3                                                           #       35         atag ccactctcgg aagta                                      - <210> SEQ ID NO 4                                                           <211> LENGTH: 543                                                             <212> TYPE: PRT                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 4                                                           - Met Thr Trp Ala Tyr Ala Ala Gly Val Pro Le - #u Ala Ser Asp Arg Tyr         #                15                                                           - Gly Leu Leu Ala Phe Gly Leu Tyr Gly Ala Ph - #e Leu Ser Ala His Leu         #            30                                                               - Val Ala Gln Ser Leu Phe Ala Tyr Leu Glu Hi - #s Arg Arg Val Ala Ala         #        45                                                                   - Ala Ala Arg Gly Pro Leu Asp Ala Ala Thr Al - #a Arg Ser Val Ala Leu         #    60                                                                       - Thr Ile Ser Ala Tyr Gln Glu Asp Pro Ala Ty - #r Leu Arg Gln Cys Leu         #80                                                                           - Ala Ser Ala Arg Ala Leu Leu Tyr Pro Arg Al - #a Arg Val Arg Val Leu         #                95                                                           - Met Val Val Asp Gly Asn Arg Ala Glu Asp Le - #u Tyr Met Val Asp Met         #           110                                                               - Phe Arg Glu Val Phe Ala Asp Glu Asp Pro Al - #a Thr Tyr Val Trp Asp         #       125                                                                   - Gly Asn Tyr His Gln Pro Trp Glu Pro Ala Al - #a Ala Gly Ala Val Gly         #   140                                                                       - Ala Gly Ala Tyr Arg Glu Val Glu Ala Glu As - #p Pro Gly Arg Leu Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Glu Ala Leu Val Arg Thr Arg Arg Cys Va - #l Cys Val Ala Gln Arg         #               175                                                           - Trp Gly Gly Lys Arg Glu Val Met Tyr Thr Al - #a Phe Lys Ala Leu Gly         #           190                                                               - Asp Ser Val Asp Tyr Val Gln Val Cys Asp Se - #r Asp Thr Arg Leu Asp         #       205                                                                   - Pro Met Ala Leu Leu Glu Leu Val Arg Val Le - #u Asp Glu Asp Pro Arg         #   220                                                                       - Val Gly Ala Val Gly Gly Asp Val Arg Ile Le - #u Asn Pro Leu Asp Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Trp Val Ser Phe Leu Ser Ser Leu Arg Tyr Tr - #p Val Ala Phe Asn Val         #               255                                                           - Glu Arg Ala Cys Gln Ser Tyr Phe His Cys Va - #l Ser Cys Ile Ser Gly         #           270                                                               - Pro Leu Gly Leu Tyr Arg Asn Asn Leu Leu Gl - #n Gln Phe Leu Glu Ala         #       285                                                                   - Trp Tyr Asn Gln Lys Phe Leu Gly Thr His Cy - #s Thr Phe Gly Asp Asp         #   300                                                                       - Arg His Leu Thr Asn Arg Met Leu Ser Met Gl - #y Tyr Ala Thr Lys Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Ser Arg Ser Arg Cys Tyr Ser Glu Thr Pr - #o Ser Ser Phe Leu Arg         #               335                                                           - Trp Leu Ser Gln Gln Thr Arg Trp Ser Lys Se - #r Tyr Phe Arg Glu Trp         #           350                                                               - Leu Tyr Asn Ala Leu Trp Trp His Arg His Hi - #s Ala Trp Met Thr Tyr         #       365                                                                   - Glu Ala Val Val Ser Gly Leu Phe Pro Phe Ph - #e Val Ala Ala Thr Val         #   380                                                                       - Leu Arg Leu Phe Tyr Ala Gly Arg Pro Trp Al - #a Leu Leu Trp Val Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Cys Val Gln Gly Val Ala Leu Ala Lys Al - #a Ala Phe Ala Ala Trp         #               415                                                           - Leu Arg Gly Cys Leu Arg Met Val Leu Leu Se - #r Leu Tyr Ala Pro Leu         #           430                                                               - Tyr Met Cys Gly Leu Leu Pro Ala Lys Phe Le - #u Ala Leu Val Thr Met         #       445                                                                   - Asn Gln Ser Gly Trp Gly Thr Ser Gly Arg Ar - #g Lys Leu Ala Ala Asn         #   460                                                                       - Tyr Val Pro Leu Leu Pro Leu Ala Leu Trp Al - #a Leu Leu Leu Leu Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Leu Val Arg Ser Val Ala His Glu Ala Ar - #g Ala Asp Trp Ser Gly         #               495                                                           - Pro Ser Arg Ala Ala Glu Ala Tyr His Leu Al - #a Ala Gly Ala Gly Ala         #           510                                                               - Tyr Val Gly Tyr Trp Val Ala Met Leu Thr Le - #u Tyr Trp Val Gly Val         #       525                                                                   - Arg Arg Leu Cys Arg Arg Arg Thr Gly Gly Ty - #r Arg Val Gln Val             #   540                                                                       __________________________________________________________________________

What is claimed is:
 1. An isolated DNA encoding a human originpolypeptide which comprises at least a fragment of the amino acidsequence set forth in SEQ ID NO:4 and which has hyaluronan synthaseactivity.
 2. The isolated DNA according to claim 1, which encodes thepolypeptide of SEQ ID NO:4.
 3. The isolated DNA according to claim 1,which comprises at least a fragment of the nucleic acid sequence setforth in SEQ ID NO:1.
 4. The isolated DNA according to claim 3, whichcomprises the nucleic acid sequence from position 149 to position 1777set forth in SEQ ID NO:1.
 5. An isolated DNA hybridizable with a nucleicacid complementary to the nucleic acid sequence set forth in SEQ ID NO:1 in 5×SSC containing 0.1% SDS, 5%(w/v) dextran sulfate, and 100 μg/mldenatured salmon sperm DNA at 60° C. for 12 hours, wherein said DNAencodes a polypeptide that has hyaluronan synthase activity.
 6. Theisolated DNA according to claim 5, wherein the hybrid of the isolatedDNA and the nucleic acid complementary to the nucleic acid sequence setforth in SEQ ID NO: 1 is stable after washing with 1×SSC containing 0.1%SDS at 60° C. for 15 minutes, and then with 0.5×SSC containing 0.1% SDSat 60° C. for 15 minutes.